Alkaline phosphatase (AP) staining in the developing zebrafish IO bones. (A) AP staining for IO1 appears first at 9.0 mm standard length (SL), which corresponds to the presumptive IO1 in Figure 3A–B. (B, C) AP broadens throughout IO1 and the canal. (D) IO2 shows canal-shaped AP that spreads and intensifies around canal walls and foramen for an IO with one ossification center (two centers not shown) (E). (F–H) AP in presumptive IO3 bones indicates osteoblast activity in the lateral line canal walls. (I–M) AP activity present in IO4 and IO5 shows the presence of osteoblasts before mineralization in the canal region of both presumptive bones. Scale bars are 100 μm. Color images available online at www.liebertpub.com/zeb

Alizarin Red staining shows mineralization in the developing zebrafish IO bones. (A) The presumptive IO1 bone is present before mineralization beginning at 8.5 mm SL. (B, C) Mineralization occurs around the foramen (arrows) and around canal walls. (D) The bony lateral line canal roof begins closing at 15.0 mm SL. (E) Mineralization of the bony lateral line canal roof continues in specimens up to 25.0 mm SL. (F and G) The IO2 sequence of development. At 17.0 mm SL, IO2 can appear as (F) one or (G) two slivers of bone depending on the number of ossification centers. (H, I) These slivers of bone eventually ossify into short canals that expand and, in the case of multiple slivers, join together to form one canal. The point in time when fusion occurs varies. (J) Canal walls begin to mineralize at 22.0 mm SL. (K–L) Mineralization of IO3 begins at 10.0–11.0 mm SL near the center of the presumptive bone. (M) The lateral line canal walls (double-headed arrows) begin to ossify, extending out in segmented projections (arrows). (N) Later in development, parallel canal wall segments begin to join and fuse as they continue to ossify outward (laterally) to form tall canal walls. By 16.0 mm SL, the dorsal canal wall begins to ossify closing over the roof. (O) Mineralization of IO3 continues to adulthood. (P, Q) At 11.0–16.0 mm SL, IO4 mineralization begins along the base of the canal. (R) At 17.0 mm SL, (I) the canal walls begin to form, mineralizing laterally (outward). (S) At 20.0 mm SL, an extension of the flat portion of IO4 extends out from the posteroventral end of bone and the canal roof begins to mineralize. (T) The canal roof closure is not complete by 25.0 mm SL. (U, V) Mineralization of IO5 begins around the circumference of this foramen. (W) Mineralization expands from the initial foramen area and also continues as a flat extension from the anterodorsal corner of IO5. (X) By 18.5 mm SL, the canal walls begin mineralization. (Y) Mineralization continues in the canal walls and flat extension and at 25.0 mm SL, the canal roof of IO5 is not yet closed. Scale bars are 100 μm.

The IO bones in a mature 3-year-old zebrafish. (A–J) are stained with Alizarin Red S and K is double stained. (A) IO1 viewed laterally. (B) IO1 viewed ventrally. (C) IO2 viewed laterally. (D) IO2 viewed anteriorly. (E) IO3 viewed laterally. (F) IO3 viewed dorsolaterally. (K) IO3 viewed laterally. (G, H) IO4 viewed laterally. (I) IO5 viewed laterally. (J) IO5 viewed ventrally. The IO bones from a mature zebrafish were (A–J) bone stained or (K) double stained for cartilage and bone and dissected. All bones show raised lateral line canal walls and lack complete canal roof mineralization (double-headed arrows). (K) The composition of the unmineralized canal roof can be visualized through Alcian blue staining (arrow). Scale bars are 100 μm